Obtention of haploid embryos and plants through irradiated pollen technique in squash (Cucurbita pepo L.)

The influence of gamma ray doses (25, 50, 75, 100, 200, 300 and 400 Gy) and genotypes (Eskenderany F₁, Acceste F₁, Sakiz, Urfa Yerli) on the induction of haploid embryos obtained by irradiated pollen technique was studied in squash (Cucurbita pepo L.). Different shapes and stages of embryos were derived from seeds extracted from fruits harvested 4–5 weeks after pollination. As a result of the present study, haploid embryos and haploid plants were obtained, with haploid production strongly influenced by gamma ray doses, embryo stages and genotypes. Gamma ray doses of 25 and 50 Gy gave the highest parthenogenetical response. All of the point shape, globular shape, arrow tips and stick-shaped embryos developed into haploid plants. However, only 53.8% of torpedo and 23.1% of heart-shaped embryos gave haploid plants. In contrast, cotyledon-shaped and amorphous-shaped embryos produced only diploid plantlets. The number of embryos per 100 seeds was the highest in ‘Eskenderany F₁’ and ‘Sakiz’ genotypes. After in vitro culture, a total of 93 haploid plantlets were obtained. This revised version was published online in August 2006 with corrections to the Cover Date.     

Anther culture in connection with induced mutations for rice improvement

Doubled haploids have long been recognized as a valuable tool in plant breeding since it not only offers the quickest method of advancing heterozygous breeding lines to homozygosity, but also increases the selection efficiency over conventional procedures due to better discrimination between genotypes within any one generation. Ten cultivars of japonica rice and nine cultivars of indica rice were evaluated for androgenic response. Various doses (10–50 Gy) of gamma rays were applied to investigate the effect of radiation on callus formation, green plant regeneration and the frequency of selected doubled haploid mutants. Similarly, the effects of colchicine concentration (10–200 mg/l) on callus induction, regeneration and fertility of green plants were observed. It was demonstrated that the dose of 20 Gy gamma rays and 30 mg/l concentration of colchicine have significant stimulation effect on regeneration of green plants from rice anther culture. The high frequency of observed doubled haploid mutants indicates that anther culture applied in connection with gamma rays is an effective way to improve rice cultivars. This revised version was published online in August 2006 with corrections to the Cover Date.     

Evaluation of <Emphasis Type="Italic">in vitro</Emphasis> water stress tolerance among EMS — Induced variants of banana (<Emphasis Type="Italic">Musa</Emphasis> spp., AAA), using “morphological,physiological and molecular” traits

Water stress is a serious environmental restriction to banana productivity. Hence, the objective of this study was to employ in vitro mutagenesis in selection and characterization of drought tolerant lines in banana. In vitro culture responses of ethyl methanesulphonate induced variants of banana cultivars, ‘Berangan Intan’ and ‘Berangan’ were assessed concerning morphological, physiological and molecular characteristics involving mutated shoot tips on MS medium supplemented with 30 g L⁻¹ PEG. The results showed that water stress tolerant lines could be obtained from induced variations. Variants L_2–5 and L_1–5 showed the highest number of leaves per shoot (2.37 and 2.06, respectively) and the lowest were recorded in the parental lines L_1-1 and L_2-1 (0.81 and 0.93, respectively). Fresh weight and shoot vigor rate indicated the maximum increase in the water stress tolerant lines compared with susceptible and non-mutated parental lines. L_2–5 exhibited the most increase in the chlorophyll and the most reduction in H₂O₂ and MDA contents when exposed to water stress. Under PEG treatment, proline and relative water content was enhanced in L_1–5, L_2–5, L_2–6, L_1–6, L_2–3, L_2–4, and L_1–4. RAPD analysis revealed polymorphism (18.35 and 21.48%) among variants derived from ‘Berangan Intan’ and ‘Berangan’, respectively. The amplified fragments generated by primers opc01, opc04, opa11, and opa20 observed to be specific for L_2–5 and L_1–5 as more tolerant followed by L_2–3, L_1–4, L_2–6, and L_1–6 as moderately tolerant lines against water stress. This study demonstrates the application of in vitro mutagenesis in selection of water stress tolerant lines of banana as a convenient, cheap, and rapid technique.     

Selection of a mutant from adventitious shoots formed in X ray treated cherry leaves and differentiation of standard and mutant with RAPDs

Summary An obstacle when using scions orin vitro shoots for mutation induction is the occurrence of chimeras. When adventitious shoots are formed from irradiated material these usually are derived from single cells, this leading to homohistont mutants. SincePrunus avium regenerates adventitious shoots from leaves at a low rate only (Yang & Schmidt, 1992), leaves of the interspecific cherry rootstock ‘209/1’ (P. cerasus ×P. canescens) were irradiated. ‘209/1’ regenerates adventitious shoots readily. Dosages applied were 5, 10, 20 and 40 Gy. Shoot production following 5 Gy irradiation was similar to the control. The application of 40 Gy resulted in strong damage with only few leaves regenerating. Among the adventitious shoots from leaves irradiated with 20 Gy one shoot was evident alreadyin vitro with thicker and smaller leaves having a serrate margin. It was cloned as ‘209/1-20m’. The clone stayed stable since 1990in vitro, in the greenhouse and the field. Compared with standard ‘209/1’ the mutant is very dwarf. Research to differentiate between standard ‘209/1’ and ‘209/1-20m’ was done using RAPDs. Among the decamer primer kits D, J, and T from Operon Technologies, Calif. Only primer OPJ05 (5‘CTCCATGGGG3’) differentiated between ‘209/1’ and ‘209/1-20m’. Rootstock ‘209/1’ showed one band of 2 kb additionally. This band is missing in the mutant.     

In vitro induction of tetraploids in Phlox subulata L.

Tetraploid plants of Phlox subulata L. were induced successfully by treating shoot tips in vitro with colchicine. Shoot tips excised from in vitro shoots were treated with four different concentrations of colchicine (0.005, 0.01, 0.02, 0.04%) in solid MS medium supplemented with 4.54 μM TDZ and 0.49 μM IBA for 10, 20 or 30 days, respectively. The survival rates of shoots tips were affected by the concentration of colchicine and the duration of treatment. High concentration and longer duration reduced survival of the shoot tips, but the effect of duration of colchicine was more than that of concentration. Tetraploid plants were obtained in all of the treatments, but the percentages of tetraploids varied among different treatments, from 25.0% to 75.0%. The most efficient condition for inducing tetraploids was to treat shoot tips with 0.005% colchicine for 20 days, with 30.0% survival rate of shoot tips and 6 tetraploid plants out of 10 plants examined. The rooted tetraploid plants were transplanted successfully in a solar greenhouse. Under the same growing condition, significant varieties in flower bud and flower sizes were detected between 2x and 4x plants. The flower diameters of tetraploid and diploid plants were 2.91 cm and 2.24 cm, respectively.     

Population genetics of self-incompatibility and developing self-compatible genotypes in niger (<Emphasis Type="Italic">Guizotia abyssinica</Emphasis>)

Two cucumber recombinant inbred lines (RILs) differing in plant habit were crossed and progeny self-pollinated to produce F₃ individuals upon which phenotypic selection was practiced to identify a base population which in turn underwent either two cycles of MAS or random mating without selection (RAN). MAS and RAN were practiced to produce F₄ and F₅ progeny sets. RIL, crossing parents, and F₃–F₅ progeny sets were then evaluated under replicated field conditions for fruit yield and quality (L:D and E:T) to evaluate gain from selection (ΔG). The broad-sense heritability (h ² B) over cycles (C) of selection ranged 0.22–0.45, 0.09–0.20, and 0.11–0.15 for yield, L:D, and E:T, respectively. Although one cycle of PHE selection followed by MAS was effective in conserving the performance of the traits examined during inbreeding, progeny performance during RAN fluctuated (F₄–F₅ generation; C₂). Lack of ΔG during advanced generations (F₄–F₅) of MAS was likely due to allelic fixation and/or optimized epistatic complementation.     

Effects of Agrobacterium tumefaciens-mediated transformation and tissue culture on storage lipids in Brassica napus

The variation obtained in storage fatty acids induced by the procedures of tissue culture and transformation with Agrobacterium tumefaciens was investigated and compared in rapeseed, Brassica napus, cv. Hanna. An increased variation in the fatty acid profiles was noted after tissue culture and transformation compared with plants derived directly from seeds. In the second generation of rapeseed transformants, T₂, the content of oleic acid ranged from 39–72%, 12–31% for linoleic acid and 7–16% for linolenic acid. This could be compared with the oleic acid content in the T₂ generation of tissue culture-derived plants which ranged between 47–76% and in seed-derived material where oleic acid ranged between 55–69%.In the T₃ generation the ranges in transgenic seeds were decreased but still larger than in the seed derived plants. The range in transgenic plants was 49–64% for oleic acid, 20–28% for linoleic acid and 9–18% for linolenic acid. The most extreme individuals, both highest and lowest in the common fatty acids, were found in the group of transformed plants independent of generation. The total lipid content was also affected by the two treatments and seeds with the lowest and highest lipid content were both found among the transformed plants. In conclusion, care should be taken to use proper controls when performing transformation experiments in order to distinguish variation in the fatty acid profiles induced by the transformation procedure and tissue culture treatments from the changes due to transgenic expression. This revised version was published online in July 2006 with corrections to the Cover Date.     

Cryopreservation of shoot tips of Chrysanthemum morifolium and related species native to Japan

Summary The shoot tips of Chrysanthemum morifolium (syn. Dendranthema grandiflorum) and related species native to Japan were cryopreserved using preculture for 2 days, slow cooling (0.2°C/min) until –40°C with 10% dimethyl sulphoxide and 3% glucose prior to immersion into LN₂ and rapid thawing. High survival rates were observed in 3 cultivars of chrysanthemum, 12 species and 2 interspecific hybrids, and slightly low survival rates in 3 species. The shoot regeneration rates of the frozen shoot tips varied from 9.4 to 100% depending on species. Shoot tips of chrysanthemum showed high viability even after a storage of 8 months in LN₂. The thawed chrysanthemum shoot tips grew and flowered normally in a greenhouse under natural conditions.Abbreviations BA 6-benzylaminopurine - DMSO dimethyl sulphoxide - NAA 1-naphtalenacetic acid     

Chasmogamous mutant,a novel character enabling commercial hybrid seed production in mungbean

Mungbean (Vigna radiata (L.) Wilczek) is a self-pollinating crop that displays significant hybrid vigor in seed yield of F₁ hybrids. Thus there is the possibility to use hybrid varieties as a breakthrough to raise the yield plateau of mungbean. However, hybrid mungbean seeds can only be accomplished by hand-pollination and thus commercial production is not possible. To encourage hybrid seed set, the plant breeder needs to develop characters that promote higher outcrossing rate such as open flower (chasmogamy). In this experiment, new chasmogamous mutants were induced by gamma irradiation at the rate of 100 and 200 Gy. The mutants were identified at a low rate of 0.4–0.7% in the M₂ generation of accession V1197, and observed for their purity by growing in plant-to-row in the M₃ and M₄ generations. A uniform chasmogamous line was hybridized to normal flower lines to study the inheritance of this character. All F₁ plants had normal flowers, while the F₂ plants segregated well with 3 normal : 1 chasmogamous ratio. When the F₁ was backcrossed to the chasmogamous parent, the progeny gave a ratio of 1 normal to 1 chasmogamous. Thus, chasmogamy was controlled by a single recessive gene, cha.     

Inheritance of beta-carotene-associated flesh color in cucumber (<Emphasis Type="Italic">Cucumis sativus</Emphasis> L.) fruit

The nutritional value of cucumber (Cucumis sativus L.) can be improved by the introgression of β-carotene (i.e., provitamin A and/or orange flesh) genes from “Xishuangbanna gourd” (XIS; Cucumis sativus var. xishuangbannanesis Qi et Yuan) into US pickling cucumber. However, the genetics of β-carotene content has not been clearly defined in this US market type. Thus, three previous populations derived from a US pickling cucumber (‘Addis’) × XIS mating were evaluated for β-carotene content, from which the high β-carotene inbred line (S₄), ‘EOM 402-10’, was developed. A cross was then made between the US pickling cucumber inbred line ‘Gy7’ [gynoecious, no β-carotene, white flesh; P₁] and ‘EOM 402-10’ [monoecious, possessing β-carotene, orange flesh; P₂] to determine the inheritance of β-carotene in fruit mesocarp and endocarp tissue. Parents and derived cross-progenies (F₁, F₂, BC₁P₁, and BC₁P₂) were evaluated for β-carotene content in a greenhouse in Madison, Wisconsin. While F₁ and BC₁P₁ progeny produced mature fruits possessing white, light-green, and green (0.01–0.02 μg g⁻¹ β-carotene) mesocarp, the F₂ and BC₁P₂ progeny mesocarp segregated in various hues of white, green, yellow (0.01–0.34 μg g⁻¹ β-carotene), and orange (1.90–2.72 μg g⁻¹ β-carotene). Mesocarp and endocarp F₂ segregation adequately fit a 15:1 [low-β-carotene (0.01–0.34 μg g⁻¹): high-β-carotene (1.90–2.72 μg g⁻¹)] and 3:1 (low-β-carotene: high-β-carotene) ratio, respectively. Likewise, segregation of carotene concentration in mesocarp and endocarp tissues in BC₁P₂ progeny adequately fit a 3:1 (low-β-carotene: high-β-carotene) and 1:1 (low-β-carotene: high-β-carotene) ratio, respectively. Progeny segregations indicate that two recessive genes control the β-carotene content in the mesocarp, while one recessive gene controls β-carotene content in the endocarp. Single marker analysis of F₂ progeny using the carotenoid biosynthesis gene Phytoene synthase determined that there was no association between this gene and the observed β-carotene variation in either fruit mesocarp or endocarp.     

Genetic parameters for yield in macadamia

Broad-sense heritability, and genetic and phenotypic correlations were estimated for yield (nut-in-shell; kernel yield; and net present value of crop) per tree in macadamia to 10 years after planting from a trial of 40 cultivars replicated over 4locations. In addition, canopy width at 10years of age was measured and used to calculate yield efficiency as yield per square metre of projected canopy area. Stem girth above the graft union was also measured. There was a low broad-sense heritability (0.06 < H <0.22) and correlation of cultivars across locations (r _gloc = 0.14–0.52) for yield per tree. However, the genetic correlation of cumulative yield was high among 3 of the 4locations (g ≥ 0.75), suggesting the higher genetic variance at Rockhampton was the main cause of the genotype by environmental interaction. Heritability was higher for canopy width (H = 0.28) and yield efficiency (H ≈ 0.47) and cultivar performance was highly correlated across locations (r _gloc ≈ 0.70) for these traits. There was a strong genetic correlation (r _g ≥ 0.90) between cumulative yield per tree of cultivars to 7 years and cumulative yield per tree to 10 years. Genetic correlation among all measures of yield per tree were high (r _g ≥ 0.73), but there was no genetic correlation with canopy width. On the other hand, small cultivars tended to have higher yield efficiency (r _g <–0.62). There was also a slight negative correlation between nut-in-shell yield per tree and kernel recovery (r _g = –0.37). These results suggest the use of family information and index selection may improve the efficiency of selection and breeding programs in macadamia. This revised version was published online in July 2006 with corrections to the Cover Date.     

Genetics of some in vitro characters in pearl millet

Summary Genetics of four in vitro characters was studied using immature inflorescence-derived callus from four inbred lines. The number of genes controlling total callus quantity varied from 5–11, those for embryogenic(E) callus quantity from 4–7, for callus growth rate from 3–12 and those for regeneration frequency varied from 4–9 in the three segregating F₂ populations. Callus derived from the dwarf plants (d₂ d₂) was phenotypically distinguishable from that of the tall accessions. The association between d₂ locus and the in vitro characters was analysed using three different approaches. It is suggested that d₂ locus might be closely linked to majority of the loci governing E callus production.     

Genetic mapping of AFLP markers in Japanese bunching onion (<Emphasis Type="Italic">Allium fistulosum</Emphasis>)

Summary The first genetic linkage map of Japanese bunching onion (Allium fistulosum) based primarily on AFLP markers was constructed using reciprocally backcrossed progenies. They were 120 plants each of (P₁)BC₁ and (P₂)BC₁ populations derived from a cross between single plants of two inbred lines: D1s-15s-22 (P₁) and J1s-14s-20 (P₂). Based on the (P₂)BC₁ population, a linkage map of P₁ was constructed. It comprises 164 markers – 149 amplified fragment length polymorphisms (AFLPs), 2 cleaved amplified polymorphic sequences (CAPSs), and 12 simple sequence repeats (SSRs) from Japanese bunching onion, and 1 SSR from bulb onion (A. cepa) – on 15 linkage groups covering 947 centiMorgans (cM). The linkage map of P₂ was constructed with the (P₁)BC₁ population and composed of 120 loci – 105 AFLPs, 1 CAPS, and 13 SSRs developed from Japanese bunching onion and 1 SSR from bulb onion – on 14 linkage groups covering 775 cM. Both maps were not saturated but were considered to cover the majority of the genome. Nine linkage groups in P₂ map were connected with their counterparts in P₁ map using co-dominant anchor markers, 13 SSRs and 1 CAPS.     

Cryopreservation of axillary shoot tips of in vitro-grown grape (Vitis) by a two-step vitrification protocol

Axillary shoot tips of in vitro grape(Vitis vinifera L. cv. CabernetSauvignon) were successfully cryopreservedby vitrification. Axillary shoot tipswere excised from 4- and 5-month oldplantlets were cultured onsolidified 1/2 MS medium with 1 mg l^-1BA at 25 ^°C. Shoot tips wereprecultured on solidified mediumsupplemented with 0.3 M sucrose for 3 daysand then treated with a mixture of 2 Mglycerol plus 0.4 M sucrose (LS solution)for 20 min at 25 ^°C. They were thendehydrated with PVS2 for 80 min at0 ^°C before being plunged into LN for1 hr. Samples were then warmed rapidly inwater at 40 ^°C. The recovery of theshoot tips amounted to approximately 60%.To enhance further recovery, a two-stepdehydration procedure by vitrification wasexamined. Osmo-protected shoot tips werefirst dehydrated with a 50% PVS2 (halfstrength of the solution) for 30 min,followed by PVS2 for 50 min at 0 ^°C.This revised dehydration procedure improvedshoot recovery from 60 to 80%. Thisprocedure by vitrification was applied toten other species/cultivars of Vitiswith an average recovery of 64%. Thismethod incorporates a simple and reliableapproach for providing high levels ofgrowth recovery. It also appears promisingfor the cryopreservation of Vitisgermplasm.     

Inheritance of fruit yield in two watermelon populations in North Carolina

Elite cultivars of watermelon (Citrullus lanatus (Thunb.) Matsum. & Nakai) are high in fruit quality but may not be the highest yielders. The objective of this study was to estimate the heritability of, and genotypic and phenotypic correlations among, yield traits in two watermelon populations developed from crosses between obsolete cultivars with high yield and elite modern cultivars. Field trials were conducted at two locations in North Carolina (Clinton and Kinston). The data were analyzed by regressing S_0:1 progeny data on S₀ parent data to estimate narrow-sense heritability. Narrow-sense heritability estimates were low for all traits measured [total fruit weight (0.04–0.12), marketable fruit weight (0.06–0.15), total fruit number (0.04–0.16), fruit size (0.18–0.19), and percent culls (0.02–0.09) in North Carolina Watermelon 1 (NCWP1) and North Carolina Watermelon 2 (NCWP2) populations, respectively]. Estimates of broad-sense heritability were higher than estimates of narrow-sense heritability. Total fruit weight and marketable fruit weight were highly correlated (r _g = 0.97–1.00). Marketable fruit weight and fruit size used as single selection criteria in NCWP1 and total fruit number in NCWP2, were predicted to give the best correlated response for total fruit weight. Narrow-sense heritability was low for fruit yield; therefore, watermelon breeders should select based on replicated progeny rows in multiple environments to maximize gain.     

Improving ovary and embryo culture techniques for efficient resynthesis of <Emphasis Type="Italic">Brassica napus</Emphasis> from reciprocal crosses between yellow-seeded diploids <Emphasis Type="Italic">B. rapa</Emphasis> and <Emphasis Type="Italic">B. oleracea</Emphasis>

The primary aim of this study was to optimize in vitro culture protocols to establish an efficient reproducible culture system for different Brassica interspecific crosses, and to synthesize yellow-seeded Brassica napus (AACC) for breeding and genetical studies. Reciprocal crosses were carried out between three B. rapa L. ssp. oleifera varieties (AA) and five accessions of B. oleracea var. acephala (CC). All the parental lines were yellow-seeded except one accession of B. oleracea. Hybrids were obtained through either ovary culture from crosses B. rapa × B. oleracea, or embryo culture from crosses B. oleracea × B. rapa. A higher rate of hybrid production was recorded when ovaries were cultured at 4–7 days after pollination (DAP). Of different culture media, medium E (MS with half strength macronutrients) showed good response for ovaries from all the crosses, the highest rate of hybrid production reaching 45% in B. rapa (1151) × B. oleracea (T₂). In embryo culture, the hybrid rate was significantly enhanced at 16–18 DAP, up to 48.1% in B. oleracea (T₃) × B. rapa (JB₂). The combinations of optimal DAP for excision and media components increased recovery of hybrids for ovary and embryo culture, and constituted an improved technique for B. rapa × B. oleracea crosses. In addition, yellow seeds were obtained from progenies of two crosses, indicating the feasibility of developing yellow-seeded B. napus through the hybridization between yellow-seeded diploids B. rapa and B. oleracea var. acephala.     

Stay green trait: variation, inheritance and its association with spot blotch resistance in spring wheat (Triticum aestivum L.)

One thousand four hundred and seven spring wheat germplasm lines belonging to Indian and CIMMYT wheat programs were evaluated for stay green (SG) trait and resistance to spot blotch caused by Bipolaris sorokiniana during three consecutive crop seasons, 1999–2000, 2000–2001 and 2001–2002. Disease severity was recorded at six different growth stages beginning from tillering to late milk stage. SG trait was measured by following two approaches: difference for 0–9 scoring of green coloration (chlorophyll) of flag leaf and spike at the late dough stage (GS 87) and a new approach of leaf area under greenness (LAUG). Germplasm lines showed a wide range (7–89) for LAUG and were grouped into four viz., SG, moderately stay green, moderately non-stay green and non-stay green (NSG). However, very few (2.2%) lines showed high expression of SG trait, i.e., LAUG >60. LAUG appeared to be a better measure of SG trait than a 0–9 scale. Mean spot blotch ratings of SG genotypes were significantly lower than those of NSG genotypes at all growth stages. Two spot blotch resistant genotypes (Chirya 3 and Chirya 7) having strong expressions of SG trait were crossed with NSG, spot blotch susceptible cv. Sonalika. Individually threshed F₂ plants were used to advance the generations. SG trait and spot blotch severity were recorded in the parents and F₁, F₃, F₄, F₅, F₆ and F_6–7 generations under disease-protected and inoculated conditions. SG trait in the F₁ generation was intermediate and showed absence of dominance. Evaluation of progenies (202–207) in the segregating generations revealed that SG trait was under the control of around four additive genes. Lines homozygous for SG trait in F₄, F₅, F₆ and F_6–7 generations showed significantly lower mean area under disease progress curve (AUDPC) for spot blotch than those with NSG expression. A positive correlation (0.73) between SG trait and AUDPC further indicated a positive influence of SG on severity of spot blotch. The study established that variation for SG trait exists in spring wheat; around four additive genes control its inheritance in the crosses studied and there is positive association between SG trait and resistance to spot blotch.     

Production and characterization of amphihaploid hybrids between <Emphasis Type="Italic">Nicotiana wuttkei</Emphasis> Clarkson et Symon and <Emphasis Type="Italic">N. tabacum</Emphasis> L

Nicotiana wuttkei Clarkson and Symon discovered in the 1990s in Australia may be of potential interest to breeders as it carries resistance to Peronospora hyoscyami de Bary. The crossability between N. wuttkei (2n = 4x = 32) and three N. tabacum (2n = 4x = 48) cultivars (‘Puławski 66’, ‘Wiślica’ and ‘TN 90’) and the morphology and cytology of their amphihaploid hybrids (2n = 4x = 40) were studied. Seeds were produced only when N. wuttkei was used as the maternal parent, but under normal germination all seedlings died. Viable F₁ hybrids of N. wuttkei × N. tabacum cv. ‘Puławski’ and N. wuttkei × N. tabacum cv. ‘Wiślica’ were obtained only by in vitro cotyledon culture. The amphihaploid plants were intermediate between the parents for most morphological traits. In 46.4% of the PMC’s, only univalents were present. The remainder of the cells had 1–5 bivalents and 1–2 trivalents. In spite of a detectable frequency of monads (2.6%), dyads (2.6%) and triads (4.5%), the hybrids were self and cross sterile.     

Non-random gametoclonal variation in rice regenerants from callus subcultured for a prolonged period under high osmotic stress

Gametoclonal variations were investigated in rice plants regenerated from anther-derived callus after subculture for two different periods (4–6 and 20 weeks) in medium with two levels (292 and 438 mM) of mannitol. A high osmotic stress by mannitol in subculture medium contributed to the maintenance of high regeneration ability. The frequency of diploid regenerants (R₀ plants) increased and that of haploids decreased from callus subcultured for the longer period in medium with the higher concentration of mannitol. R₁ regenerant lines displayed variations in all six agronomical traits studied at the frequencies of 63% and 88% after subculture for 4–6 weeks and 20 weeks, respectively. High homozygosity was suggested in most of the R₁ lines for these traits by the similar levels of variance to those in the control variety. A principal component analysis showed a skewed distribution of variants towards dwarfness and less panicle weight in the regenerants from callus subcultured for 20 weeks with 438 mM mannitol. Two short culmn variants were shown to be due to single or double recessive mutations. The results suggested that prolonged subculture under high osmotic stress causes non-random selection of homozygous gametoclonal variations in rice anther culture. This revised version was published online in August 2006 with corrections to the Cover Date.     

Molecular markers linked to <Emphasis Type="Italic">Bn</Emphasis>;<Emphasis Type="Italic">rf</Emphasis>: a recessive epistatic inhibitor gene of recessive genic male sterility in <Emphasis Type="Italic">Brassica napus </Emphasis>L.

7–7365AB is a recessive genic male sterile (RGMS) two-type line, which can be applied in a three-line system with the interim-maintainer, 7–7365C. Fertility of this system is controlled by two duplicate dominant epistatic genes (Bn;Ms3 and Bn;Ms4) and one recessive epistatic inhibitor gene (Bn;rf). Therefore an individual with the genotype of Bn;ms3ms3ms4ms4Rf_ exhibits male sterility, whereas, plant with Bn;ms3ms3ms4ms4rfrf shows fertility because homozygosity at the Bn;rf locus (Bn;rfrf) can inhibit the expression of two recessive male sterile genes in homozygous Bn;ms3ms3ms4ms4 plant. A cross of 7–7365A (Bn;ms3ms3ms4ms4RfRf) and 7–7365C (Bn;ms3ms3ms4ms4rfrf) can generate a complete male sterile population served as a mother line with restorer in alternative strips for the multiplication of hybrid seeds. In the present study, molecular mapping of the Bn;Rf gene was performed in a BC₁ population from the cross between 7–7365A and 7–7365C. Bulked segregant analysis (BSA) and amplified fragment length polymorphism (AFLP) technique was used to identify molecular markers linked to the gene of interest. From a survey of 768 primer combinations, seven AFLP markers were identified. The closest marker, XM5, was co-segregated with the Bn;Rf locus and successfully converted into a sequence characterized amplified region (SCAR) marker, designated as XSC5. Two flanking markers, XM3 and XM2, were 0.6 cM and 2.6 cM away from the target gene, respectively. XM1 was subsequently mapped on linkage group N7 using a doubled-haploid (DH) mapping population derived from the cross Tapidor × Ningyou7, available at IMSORB, UK. To further confirm the location of the Bn;Rf gene, additional simple sequence repeat (SSR) markers in linkage group N7 from the reference maps were screened in the BC₁ population. Two SSR markers, CB10594 and BRMS018, showed polymorphisms in our mapping population. The molecular markers found in the present study will facilitate the selection of interim-maintainer.